One of the most important activities of modern medical and biochemical fields is conducting medical diagnostic assays, such as cell culture assays, immunoassays, DNA hybridization assays, robot assisted sample handling processes, and microfluid sample processing. These activities permit safe and effective medical diagnoses, as well as thorough and accurate biochemical investigations and research.
Automated microbial culturing systems have been developed in recent years to test for a variety of diseases in a clinical laboratory setting. These systems often include culture tubes containing samples, selective growth media, and a fluorescent indicator that responds to the growth of the microorganisms. The tubes are continually processed by an optical reader that measures changes in fluorescent properties of the sample in order to detect such microbes as tuberculosis or antibiotic resistant Staph. aureus. Unfortunately, such systems can take days to culture a sufficient quantity of the microbes necessary for identification with standard test methods.
Biocards, such as those described in U.S. Pat. No. 5,609,828, have also been developed to carry out multiple assays from a single sample extracted from blood, fluids, or other tissue of a patient. These samples are usually examined using spectroscopic or other automated analysis techniques. Biocards are typically molded in plastic and are designed to receive a liquid sample into a series of small sample wells formed in the card. Each sample well normally contains a different set of dried reagent (selective growth nutrients and indicator dies) for identifying different biological agents within the sample. During analysis, the sample enters an intake port, collects in an intake reservoir, and travels along distribution channels to the sample wells. Each sample well also typically includes a bubble trap designed to trap gases formed by growing microorganism colonies. The reagents within the sample wells dissolve when the fluid sample is introduced. After incubation of the sample in the sample wells, a card reader performs automated spectroscopic or fluorescence analysis on each well. Although analysis with biocards can be successful, analysis times are quite long unless the microorganisms are first cultured to increase their number. In addition, closely related strains of microorganisms are hard to differentiate by these methods.
Efforts have been made to develop assay techniques for the analysis of nucleic acids and proteins that shorten the delay associated with culture techniques, increase the specificity of the assays, and provide means for detecting new diseases. One such effort has been the development of DNA amplification technologies that provide a means to produce hundreds of millions of copies of a selected DNA target in less than one hour. Microorganisms of interest are first lysed to release their DNA material. The DNA material is isolated and then treated with reagents to perform an amplification of an oligonucleotide sequence specific to the microorganism of interest. While polymerase chain reaction (PCR) is the most well known of these amplification methods, it requires temperature cycling and continued reagent additions. Other methods, such as a strand displacement amplification approach developed at Becton Dickinson of Franklin Lakes, N.J., can be performed in a single sample well in 15 minutes at constant temperature. Such amplification methods help to overcome problems related to complexity and sensitivity in genomic DNA analysis.
Once the DNA target has been “amplified” by reproduction to produce numerous amplicons, complementary oligonucleotide probes can be used to capture the DNA amplicons. These probes selectively retain the DNA amplicon, allowing them to be isolated and identified. However, these probes have traditionally relied upon diffusion controlled processes to capture the DNA amplicons. Diffusion can take hours to complete, and is a significant hurdle to rapid identification of the DNA target. Although such diffusion methods are significantly quicker than prior cell culture techniques, they are still relatively slow compared to the rapid rate of DNA amplification.
An alternative to DNA amplification, known as the Southern Blot, involves cleaving the DNA with restrictive enzymes, separating the DNA fragments on an electrophoresis gel, blotting to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the crude DNA sample, thereby helping to improve the hybridization specificity and sensitivity. However, the total number of targets generated in a Southern Blot is far less than the number of amplicons generated by DNA amplification methods. In addition, the electrophoretic separation can take hours to complete.
Recently, efforts have been made to hasten the separation and capture of DNA amplicons. Researchers have developed micro-electrode arrays that speed up the capture process by using free-field electrophoresis to concentrate and purify target DNA on the individual probes of the array. These arrays create a charged electrical field that isolates the charged DNA amplicons at one or more probes on the array. This type of microelectronic array, known as an “addressable programmable electronic matrix” (“APEX”), can reduce the time to perform the capture process from hours to minutes. A number of patents describe silicon based chips having APEX arrays. For example, U.S. Pat. Nos. 5,653,939 and 5,632,957 teach the manufacture of rigid silicon APEX arrays using a lithographic process. Although these silicon APEX arrays permit enhanced capture rates, they are relatively expensive to produce. Also, they cannot be easily reused without being cleaned and repatterned with DNA probes in a manufacturing environment. Even though they are relatively expensive, the silicon APEX arrays are typically used once and then discarded.
Thus, existing APEX arrays have improved the speed of performing DNA identification tests, but have failed to address the need for cost-effective, mass manufacturable APEX assay systems. In view of the significant expense associated with existing APEX chip systems, a need exists for an APEX chip that provides the increased speed of a programmable microelectronic matrix, but can be done for less expense than existing silicon-based chips.